The Notch signaling pathway is an evolutionarily conserved mechanism that plays a fundamental role in cell fate decisions and a number of developmental cascades in vertebrate and invertebrate tissues. Notch is a glycoprotein presented on the cell surface as a large, transmembrane, heterodimeric protein, and functions as a signal transduction receptor.

The biosynthesis of Notch begins in the cell's endoplasmic reticulum, where Notch initially is a single polypeptide chain (about 300 kDa) and undergoes folding. Then Notch is cleaved in the tans-Golgi network by a furin-like convertase into an extracellular domain of about 200 kDa and an intracellular domain of about 110 kDa. This cleavage is an essential event in the formation of functional Notch protein. Notch has a large extracellular domain composed mainly of 36 tandem epidermal growth factor-like (EGF) modules and several are predicted sites of O-linked fucosylation.

O-linked fucose saccharides modify specific EGF sites at a consensus sequence (C2XXGGS/TC3 or C2X3-5S/TC3) between the second and third evolutionarily conserved cysteines of the EGF module. O-fucose modifies the amino acids serine and threonine, each which contains a hydroxyl group. The elongation of O-fucose saccharides by Fringe, an O-fucose-b1,3-N-acetylglucosaminyltransferase, has been shown to modulate the Notch signaling pathway. The presence of O-fucose on the Notch receptor has raised the question of whether the absence of O-fucose at a particular EGF site will affect the expression of Notch or the efficiency of the production of Notch in mammalian cells (Chinese hamster ovary Lec1 cells). As a primary step to begin addressing the role of the O-fucose glycans on the Notch receptor, several mutations in predicted O-fucose glycosylation sites on mouse Notch1 have been generated. Serine or threonine residues in the consensus sequences have been converted to alanine, thus eliminating the presence of O-fucose. Then the mutations on the full length mouse Notch1 gene were transfected into mammalian cells and were analyzed for expression of Notch. Results are still pending. This project was supported by a grant from the National Institutes of Health to Dr. R. Haltiwanger (GM61126).